Figure 2

Manipulation of NOS is sufficient to mimic activity-manipulation in developing embryos. (A) Electroshock of L3 larvae showed a significant increase in RT in NOS mutants (Nos¹ and NosΔ^all). One-way ANOVA (F_{(3, 106)} = 44.73, p < 0.0001) followed by Bonferroni’s post-hoc test, n = 20 in control, Canton S, n = 30 in the other groups). The hemizygotic CRISPR mutant ((NosΔ^all/CyO, DGY) exhibited no increase in RT: 116 ± 5 s, p = 0.6104). (B) Genetic downregulation of NOS (NOS^RNAi) or overexpression of macNOS are each sufficient to increase RT (elav^C155 > NOS^RNAi: 154 ± 8 s, elav^C155 > macNOS: 166 ± 8 s vs. CTRL: 101 ± 8 s). One-way ANOVA (F_(2,87) = 16.55, p < 0.001) followed by Bonferroni’s post-hoc test, ***p < 0.0001, n = 30 in each group. (C) Whole-cell recordings of endogenous SRCs from L3 aCC following pan-neuronal (elav^C155) genetic manipulation of NOS. (D) Knock-down (NOS^RNAi, n = 10) or overexpression of macNOS (n = 10) significantly increased SRC duration. GFP^RNAi was used as control (n = 13). One-way ANOVA (F_{(2, 30)} = 22.43, p < 0.0001) followed by Bonferroni’s post-hoc test, ***p < 0.0001. (E) Temporal regulation of macNOS expression in motoneurons (OK6 > Gal$) was achieved through GAL80^ts (“ + ” or “ − ” shows macNOS expression to be activated or suppressed, respectively). GAL4-mediated expression of macNOS during embryogenesis, but not during larval stage, led to an increase in synaptic current duration at L3. One-way ANOVA (F_(3, 29) = 16.36, p < 0.001) followed by Bonferroni’s post-hoc test, ***p < 0.001, ***p = 0.0021. Dotted lines represent reference values obtained from OK6 > macNOS and, as a further control, the UAS-macNOS parental line. (F) Under identical temperature-controlled conditions, the expression of GFP^RNAi, used as an additional control, did not show detectable change in synaptic current duration (n = 10 in each group).